Glycine anhydride dimethylol as a biocide and preservative

ABSTRACT

The use of glycine anhydride dimethylol, also known as 1,4-bis(hydroxymethyl)-2,5-piperazinedione, comprising the formula ##STR1## to kill microorganisms present in or on a host substance and to preserve products susceptible to contamination by a variety of microorganisms is disclosed.

This application is a Division of application Ser. No. 07/694,252, filedMay 1, 1991 now U.S. Pat. No. 5,244,653.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

This invention relates to the use of glycine anhydride dimethylol, as abiocide and an antimicrobial agent to kill microorganisms present in oron substances and to preserve a variety of substances susceptible tomicrobial contamination.

2. Description of the Prior art

Glycine anhydride dimethylol, also known as1,4-bis(hydroxymethyl)-2,5-piperazinedione is a known chemical compoundprepared by reacting glycine anhydride, also referred to as2,5-piperazinedione with formaldehyde according to the followingformula: ##STR2## See, e.g. Cherbuliez et al., Helv. Chim. Octa, 5, 678,688 (1923).

SUMMARY OF THE INVENTION

It is an object of the invention to provide a method for killingmicroorganisms present in or on a host substance by administering to thehost an effective biocidal amount of glycine dimethylol anhydride.

It is a further object of the invention to provide a method forpreserving products susceptible to contamination by microorganisms byincorporating into the product an antimicrobial effective amount ofglycine anhydride dimethylol.

It is a further object of the invention to provide preserved products,that is products which have been protected against contamination bymicroorganisms, containing glycine anhydride dimethylol in an amountwhich is effective to preserve the product against contamination bymicroorganisms.

Other objects and advantages of the invention will be evident to thoseof skill in the art upon review of the detailed description whichfollows.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is based upon the discovery that glycine anhydridedimethylol, also known as 1,4-bis(hydroxymethyl)-2,5-piperazinedione,represented by the formula: ##STR3## exhibits biocidal activity againsta wide range of microorganisms, including bacteria, yeast and mold. Thiscompound, therefore, is an effective biocidal agent which killsmicroorganisms present in or on a host substance and inhibits microbialgrowth in or on a variety of substances and products, whether they besolid, liquid or emulsion form. Such products include, but are notlimited to, cosmetics, foodstuffs, pharmaceuticals, paints, cutting oilsor fluids, agricultural products, oil drilling fluids, lubricants, paperindustry products, embalming solutions, cold sterilized medical anddental equipment, cooling towers, fabric impregnation, latexes, swimmingpools, inks, household disinfectants, waxes and polishes, toilet bowlcleaners, bathroom cleaners, laundry detergents, soaps, woodpreservatives, hospital and medical antiseptic and adhesives. Of theseproducts, foodstuffs, pharmaceuticals, cosmetic and personal hygieneproducts such as shampoos are of particular interest.

Other uses of glycine anhydride dimethylol according to the inventioninclude industrial uses where the compound is used to control microbialgrowth, such as for slime control in the effluent of paper mills.

By virtue of its biocidal activity, glycine anhydride dimethylol can beused to kill microorganisms which have contaminated a host substance oras a preservative to inhibit and prevent growth of microorganisms insubstances susceptible to microbial growth. The compound is alsoeffective in disinfecting and/or sterilizing surfaces prone tocontamination by microorganisms. The effective amount of glycineanhydride dimethylol required will, of course, vary depending upon themode of use, e.g., as a biocidal agent to kill an established colony ofmicroorganism or as a preservative to inhibit growth of microorganisms,to which it is directed. Those of skill in the art will have nodifficulty in determining the requisite effective amounts for particularuses.

When treating a host substance, the glycine anhydride dimethylol can beadministered thereto as a solid powder, in aqueous solution or in anemulsion alone or with other ingredients, including other biocidesand/or medicaments. The amount of glycine anhydride dimethylol requiredto be incorporated into a product for preservation will also varydepending upon the product being protected and the environment of use.Generally, products containing less than about 1.0% by weight of glycineanhydride dimethylol will be effectively protected from microbialcontamination in most environments. Principally for economic reasons,concentrations less than about 0.5% by weight are preferred. Those ofskill in the art will have little difficulty in selecting specificamounts required for particular uses based upon the informationcontained herein, especially in the examples.

In addition to glycine anhydride dimethylol, other conventionalingredients may be incorporated into the product requiring preservation,including other antimicrobial agents, suspending agents, wetting agents,anti-scaling agents, corrosion inhibitors and pH control agents. Theselection of the particular addition ingredients, if any, will dependupon the particular product requiring preservation.

When blending with the product to be preserved, the glycine anhydridedimethylol may be incorporated as a solid powder or in aqueous solution.The powder is the preferred form since it is easier to handle than aliquid. When the application calls for a liquid formulation, the powdercan be readily dissolved in aqueous media. The glycine anhydridedimethylol may be blended into the product as the sole antimicrobialagent or as part of an antimicrobial system with other antimicrobialagents. Such systems are particularly effective when it is necessary topreserve a product against a wide range of microorganisms. By carefulselection of the antimicrobial agents in the system, antimicrobialactivity against a particular group of microorganisms can be optimizedfor a particular product. When using an antimicrobial system it isfurther advantageous to incorporate the antimicrobial agents in a commonsolvent, such as propylene glycol, which also exhibits antimicrobialactivity and so serves the dual function of solvent and antimicrobialagent.

This invention thus provides an effective means for preservingsubstances susceptible to microbial contamination by inhibiting growthof bacterial, yeast and mold. The compound of the invention is readilyincorporated into a variety of forms including liquids, solids andemulsions.

To further illustrate the various aspects of the invention, thefollowing examples are provided. However, it is to be understood thattheir purpose is entirely illustrative and in no way intended to limitthe scope of the invention. In the examples, all percentages are givenby weight unless otherwise noted.

EXAMPLE 1

Preparation of glycine anhydride dimethylol.

To a stirred slurry of 11.4 g. (0.1 mole) of glycine anhydride in 41.0ml. of water was added 16.2 g. (0.2 mole) of 37% aqueous formaldehyde.Addition of 0.10 g. of 10% aqueous sodium hydroxide solution caused thesuspended solids to dissolve, followed by formation of a heavy whiteprecipitate. The mixture was heated to 70° C., by which time all solidsdissolved. On cooling to room temperature, white crystals separated. Thecrystals were collected, washed with cold water, and dried in vacuum at100° C. for 1 hour. The white crystalline product, glycine anhydridedimethylol weighed 12.6 g. (72.4% yield), m.p. 185°-188° C. dec. Anal.Calcd. for C₆ H₁₀ N₂ O₄ (174.16): N, 16.09. Anal. Found: N, 16.17.

EXAMPLE 2

Microbiological Activity

A. Minimum Inhibitory Concentration (MIC)

Aqueous solutions of 1% glycine anhydride dimethylol were seriallydiluted with sterile distilled water, and aliquots of differentconcentrations were mixed with equal volumes of inoculated culturemedium. AOAC broth was used for the bacterial culture media, andSabourand Liquid Medium was used for the yeast and mold cultures. Themixtures of glycine anhydride dimethylol solution and culture media wereincubated at 35° C. for 48 hours for bacteria and yeast, and at 26° C.for 7 days for mold. Microbial growth or no growth were determined byvisual inspection for cloudiness. The MIC for a given microorganism wasthe lowest concentration of antimicrobial that remained clear after theincubation period. Results are as follows:

    ______________________________________                                        Bacteria                       MIC                                            ______________________________________                                        Gram-Neg.                                                                             Pseudomonas aeruginosa                                                                        ATCC 9027  310 ppm                                    Gram-Pos.                                                                             Staphylococcus aureus                                                                         ATCC 6538  310 ppm                                    Yeast   Candida albicans                                                                              ATCC 10231 310 ppm                                    Mold    Aspergillus niger                                                                             ATCC 16404 2500 ppm                                   ______________________________________                                    

B. Challenge tests on a cosmetic emulsion

A typical cosmetic emulsion was prepared for microbiological challengetesting. The emulsion was prepared in four different variations: with nopreservative added, with 0.05% glycine anhydride dimethylol, with 0.10%glycine anhydride dimethylol, and with 0.20% glycine anhydridedimethylol. The formulation used for challenge testing was as follows:

    ______________________________________                                                       % W/W                                                          ______________________________________                                        Phase A                                                                       Water, Demineralized                                                                           67.90   67.85   67.80 67.70                                  Triethanolamine 99%                                                                            1.00    1.00    1.00  1.00                                   Phase B                                                                       Laneth-5 (and) Ceteth-5 (and)                                                 Oleth-5 (and) Steareth-5                                                                       0.50    0.50    0.50  0.50                                   Mineral Oil      2.50    2.50    2.50  2.50                                   Stearic Acid, XXX                                                                              5.00    5.00    5.00  5.00                                   Cetyl Alcohol    1.00    1.00    1.00  1.00                                   Glyceryl Stearate (and)                                                                        1.50    1.50    1.50  1.50                                   PEG-100 Stearate                                                              Phase C                                                                       Citric Acid (30% Solution)                                                                     0.60    0.60    0.60  0.60                                   Phase D                                                                       Water, Demineralized                                                                           20.00   20.00   20.00 20.00                                  Glycine Anhydride                                                                              --      0.05    0.10  0.20                                   Dimethylol (GAD)                                                                               100.00  100.00  100.00                                                                              100.00                                 ______________________________________                                    

Challenge tests were carried out using four different microorganisms,and microbial counts were made after incubating 24, 48, and 72 hours.The USP Antimicrobial Preservative Effectiveness Test (UPS XXII Method)was used with minor modifications.

    ______________________________________                                        MICROBIAL COUNTS, 24 HOURS                                                             No      0.05%     0.10%   0.20%                                               Preservative                                                                          GAD       GAD     GAD                                        ______________________________________                                        P. aeruginosa                                                                            9 × 10.sup.6                                                                      <10       50    100                                      S. aureus  2 × 10.sup.6                                                                      4 × 10.sup.4                                                                      5 × 10.sup.4                                                                  2 × 10.sup.3                       C. albicans                                                                              2.5 × 10.sup.4                                                                    1 × 10.sup.4                                                                      50    <10                                      A. niger   5 × 10.sup.4                                                                      500       <10   <10                                      ______________________________________                                    

    ______________________________________                                        MICROBIAL COUNTS, 48 HOURS                                                             No      0.05%     0.10%   0.20%                                               Preservative                                                                          GAD       GAD     GAD                                        ______________________________________                                        P. aeruginosa                                                                            1 × 10.sup.6                                                                      <10       15    60                                       S. aureus  6 × 10.sup.6                                                                      100       15    200                                      C. albicans                                                                              2 × 10.sup.4                                                                      1 × 10.sup.4                                                                      <10   <10                                      A. niger   4 × 10.sup.4                                                                      <10       <10   <10                                      ______________________________________                                    

    ______________________________________                                        MICROBIAL COUNTS, 72 HOURS                                                             No      0.05%     0.10%   0.20%                                               Preservative                                                                          GAD       GAD     GAD                                        ______________________________________                                        P. aeruginosa                                                                            1 × 10.sup.8                                                                      <10       <10   <10                                      S. aureus  5 × 10.sup.8                                                                      <10       <10   <10                                      C. albicans                                                                              2 × 10.sup.4                                                                      1 × 10.sup.3                                                                      <10   <10                                      A. niger   3 × 10.sup.4                                                                      <10       <10   <10                                      ______________________________________                                    

C. Challenge tests on a shampoo

A typical shampoo was prepared both with and without glycine anhydridedimethylol, and then challenge tested with several microorganisms. TheUSP Antimicrobial Preservative Effectiveness Test. (USPXXII Method) wasused with minor modifications, with microbial counts made after 24, 48and 72 hours. The same microorganisms (same ATCC numbers) were used asnoted above.

The shampoo formulations were as follows:

    ______________________________________                                                           % W/W                                                      ______________________________________                                        Phase A                                                                       Water, Demineralized 25.00   25.00                                            Ammonium Lauryl Sulfate                                                       Cocamide DEA         6.50    6.50                                             Cocamideopropyl Botaine                                                                            5.00    5.00                                             Phase B                                                                       Citric Acid, 30% Solution                                                                          *       *                                                Phase C                                                                       Water, Deionized     20.00   20.00                                            Glycine Anhydride Dimethylol                                                                       --      0.20                                                                  100.00  100.00                                           ______________________________________                                    

Microbial challenge test results were as follows:

    ______________________________________                                        MICROBIAL COUNTS, 24 HOURS                                                                No Preservative                                                                         0.2% GAD                                                ______________________________________                                        P. aeruginosa 5 × 10.sup.5                                                                        50                                                  S. aureus     <10         <10                                                 C. albicans   2 × 10.sup.4                                                                        <10                                                 A. niger      5 × 10.sup.4                                                                        <10                                                 ______________________________________                                    

    ______________________________________                                        MICROBIAL COUNTS, 48 HOURS                                                                No Preservative                                                                         0.2% GAD                                                ______________________________________                                        P. aeruginosa 1 × 10.sup.4                                                                        <10                                                 S. aureus     <10         <10                                                 C. albicans   2 × 10.sup.4                                                                        <10                                                 A. niger      1 × 10.sup.5                                                                        <10                                                 ______________________________________                                    

    ______________________________________                                        MICROBIAL COUNTS, 72 HOURS                                                                No Preservative                                                                         0.2% GAD                                                ______________________________________                                        P. aeruginosa 50          <10                                                 S. aureus     <10         <10                                                 C. albicans   2 × 10.sup.4                                                                        <10                                                 A. niger      5 × 10.sup.3                                                                        <10                                                 ______________________________________                                    

D. Preservation of milk

The microbial spoilage of whole milk at room temperature was preventedby the incorporation of 0.16% glycine anhydride dimethylol. Thefollowing series of tests were terminated after 59 days.

    ______________________________________                                        Preservative Added                                                                         Time to Spoilage                                                 to Whole Milk                                                                              (i.e. development of odor, curdling, etc.)                       ______________________________________                                        none         10 days                                                          0.16%        59 + days                                                        0.16%        59 + days                                                        0.32%        59 + days                                                        0.32%        59 + days                                                        ______________________________________                                    

While the invention has been described in terms of certain preferredembodiments, one skilled in the art will readily appreciate that variousmodifications, changes, omissions and substitutions may be made Withoutdeparting from the spirit thereof. It is intended, therefore, that thepresent invention be limited solely by the scope of the followingclaims.

What is claimed is:
 1. A method of killing microorganisms in or on ahost substance comprising administering to the substance an effectivebiocidal amount of glycine anhydride dimethylol of the formula: ##STR4##2. The method as defined in claim 1, wherein the glycine anhydridedimethylol is administered in aqueous solution.
 3. The method as definedin claim 1, wherein the glycine anhydride dimethylol is administered inan emulsion.
 4. The method as defied in claim 1, wherein the glycineanhydride dimethylol is administered as a powder.
 5. The method asdefined by claim 1, wherein the glycine anhydride dimethylol isadministered in conjunction with another biocidal agent.